The long-term objective of the proposed study is to better understand the pathogenesis of hospital-acquired pneumonia caused by Pseudomonas aeruginosa. The general strategy is to characterize the role of the P. aeruginosa type III secretion system in hospital-acquired pneumonia. This complex secretion pathway transports and injects four known effector proteins into host cells: ExoS, ExoT, ExoU (also known as PepA), and ExoY. Interestingly, clinical isolates differ in the combination of effector proteins they secrete. Recent studies are beginning to define the role of individual effector proteins in pathogenesis. Preliminary studies using bacterial mutants indicate that ExoS, ExoT, ExoU, and ExoY all have cytotoxic effects on mammalian cells in cell culture systems. ExoT, ExoU, and possibly ExoS contribute to virulence in animal models of pneumonia. In addition, ExoU secretion is associated with worse clinical outcomes in humans with hospital-acquired pneumonia. Together, these findings support an important role for type III effector proteins in the acute pneumonia, although the exact role of each effector protein and the mechanisms by which these proteins lead to the pathophysiological consequences of pneumonia remain to be defined and are the subject of this proposal. Our preliminary data suggest that type III secretion contributes to bacterial persistence, dissemination, and mortality as well as neutrophil killing and suppression of proinflammatory cytokine release in a mouse model of pneumonia. Further defining the role of individual effector proteins in these processes is crucial to our understanding of the pathogenesis of hospital-acquired pneumonia caused by P. aeruginosa. It is hypothesized that specific effector proteins play an important role in the pathogenesis of hospital-acquired pneumonia, including the prevention of bacterial clearance by neutrophils. Furthermore, it is hypothesized that because of these effects secretion of specific effector proteins can be used as markers for strains associated with especially severe hospital-acquired pneumonia in human patients. Studies using both mice and humans will be performed to define the roles of these effector proteins in the pathogenesis of acute pneumonia, including modulation of the inflammatory response and resistance to neutrophil-mediated clearance, and to determine whether secretion of particular effector proteins serves as a marker for strains capable of causing especially severe disease in human patients with hospital-acquired pneumonia.